CN108368533A - The method for counting big visible peristalsis visible intestinal peristalsis bacterium colony - Google Patents
The method for counting big visible peristalsis visible intestinal peristalsis bacterium colony Download PDFInfo
- Publication number
- CN108368533A CN108368533A CN201680071402.8A CN201680071402A CN108368533A CN 108368533 A CN108368533 A CN 108368533A CN 201680071402 A CN201680071402 A CN 201680071402A CN 108368533 A CN108368533 A CN 108368533A
- Authority
- CN
- China
- Prior art keywords
- culture apparatus
- hydrogel
- coliform
- dairy products
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/04—Flat or tray type, drawers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/22—Transparent or translucent parts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/06—Plates; Walls; Drawers; Multilayer plates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
Abstract
The present invention provides a kind of methods that statistics is present in the aerogenesis coliform in the dairy products of culture.The method may include forming a kind of mixture, the diluent of the dairy products and predetermined volume of the mixture comprising predetermined amount；It is inoculated with film culture apparatus with the mixture to form hydrogel, the hydrogel includes nutrient medium, the lactose of effective concentration and the MOPS of effective concentration grown for coliform；The vaccinated film culture apparatus is cultivated under conditions of promoting coliform growth；And it identifies and drops into capable counting to being present in the aerogenic bacteria in the vaccinated culture apparatus.
Description
Cross reference to related applications
This application claims the priority for the U.S. Provisional Patent Application 62/263,932 for being filed on December 7th, 2015, should
The disclosure of temporary patent application, which is incorporated by reference, to be incorporated herein.
Background technology
Coliform is a kind of diversified relevant microorganism of metabolism.Category bacterium is by them by a kind of lactose (presence
Disaccharides in milk) ability of one group of metabolic by-product including carbon dioxide gas is fermented into limit.Therefore, directly
And the ability of coliform depends on the ability that detaches bacterium colony aerogenesis of the detection by bacterium in accurate geo-statistic sample.
Film culture apparatus replaces traditional microorganism statistical technique and is advantageously used, and traditional technology is using for example
Culture dish (Petri dishes) containing agar medium.Several examples of such film culture apparatus, which are described in, authorizes
In the United States Patent (USP) 4,565,783 of Hansen et al. and the United States Patent (USP) 5,364,766 for authorizing Mach et al.；They full text with
Way of reference is incorporated herein.In the exemplary device of Hansen et al. reports, gelling agent and microorganism growing nutrient object will be contained
The powder coating of the cold-water-soluble drying of matter is in waterproof substrate.Surface is coated with and contains indicating dye and powdered gelling
The transparent visible emulsion sheet of the acrylic ester adhesive of agent is attached on coated base material.
When this apparatus is used, the aqueous specimen of predetermined amount, which is normally placed in, contacts coated base material and emulsion sheet
It is placed on above sample and base material.The dry powder of aqueous specimen hydration dissolving, subsequently forms gel entrapment culture base, can tie up
Hold microorganism growth.During growth period, the indicating dye being adhered on emulsion sheet reacts in the presence of viable microbial organisms,
There is provided detectable response, it can be seen that the bacterial clump grown in culture apparatus.
(such as CO is generated in order to detect aerogenic bacteria2Coliform), the device of Hansen et al. construction preferably causes to give birth to
Object origin cause of formation gas is trapped at the aerogenesis bacterium colony in the neighbouring gel entrapment culture base between base material and emulsion sheet.
Invention content
The present disclosure relates generally to the methods for cultivating and counting the microorganism for belonging to coliform.In addition, the disclosure relates to
And the device for counting the microorganism in sample.Specifically, this disclosure relates to be detected in film culture apparatus and count production
The ameliorative way of gas coliform.
The method that the disclosure provides the aerogenesis coliform that statistics is present in the dairy products of culture.This method may include being formed
A kind of mixture, the mixture include the diluent of the dairy products and predetermined volume of predetermined amount；It is inoculated with film with the mixture
Culture apparatus to form hydrogel, the hydrogel include the nutrient medium grown for coliform, the lactose of effective concentration and
The MOPS of effective concentration；Vaccinated film culture apparatus is cultivated under conditions of being conducive to coliform growth；And identification is simultaneously
Capable counting is dropped into being present in the aerogenic bacteria in vaccinated culture apparatus.
In any embodiment of this method, diluent may include MOPS.In any embodiment, in diluent
The concentration of MOPS can be >=20mM and≤50mM.
Word " preferred " and " preferably " refer to the embodiment party of the present invention that certain advantageous effects can be provided in some cases
Case.However, under identical circumstances or it is other in the case of, other embodiments are alternatively preferably.In addition, to one or more
The statement of preferred embodiment does not imply that other embodiments are disabled, and is not intended to exclude other embodiments
Outside the scope of the present invention.
As used herein, " a kind of (a) ", " (be somebody's turn to do) ", " at least one (a) " and " one kind is (a) or a variety of
(a) " is used interchangeably.Thus, for example, the method for detection "an" microorganism can be taken to mean that this method detectable " one
Kind is a variety of " microorganism.
Term "and/or" means any two of one or all or listed elements of listed elements or more
Combination.
In addition, herein, the numberical range stated by endpoint is comprising all numerical value contained within the scope of this (for example, 1
Include 1,1.5,2,2.75,3,3.80,4,5 etc. to 5).
As used herein, " film culture apparatus " refers to a kind of culture apparatus comprising is attached on sheet emulsion sheet
Sheet substrate, and base material and emulsion sheet respectively have main surface inwardly and outwardly.The table main inwardly of base material and/or emulsion sheet
Face is the coating substantially free of water, and it includes cold-water-soluble gelling agents.Optionally, base material and/or emulsion sheet be inwardly
Main surface has the coating substantially free of water, and it includes the nutrient mediums being adhered thereto.Optionally, nutrient medium
And/or gelling agent can be adhered on adhesive phase, which is coated on base material and/or emulsion sheet.Optionally, film culture apparatus
It may also include slim trepanning spacer, adhere in the inward faces of base material or emulsion sheet.Film culture apparatus it is unrestricted
Property example is disclosed in United States Patent (USP) 4,565,783；5,089,413；5,137,812；In 5,232,838；They full text with
Way of reference is incorporated herein.
As used herein, " dairy products " refer to the product containing and/or from milk.As used herein, " the breast system of culture
Product " refer to that the milk food through lactobacillus-fermented, the lactic acid bacteria have for example belonged to lactobacillus (Lactobacillus), breast
The bacterium of Coccus (Lactococcus) or Leuconostoc (Leuconostoc).The dairy products of culture include but not limited to
Buttermilk, sour cream, cheese, white soft junket, Yoghourt and the yogurt of culture.
As used herein, " MOPS buffer solutions ", " MOPS diluents ", " diluent for including MOPS " and " comprising MOPS
Aqueous solution " refers to the composition for including (3- (N- morpholines) propane sulfonic acid).When being adjusted to specific pH, " MOPS buffer solutions ",
" MOPS diluents " or " aqueous solution for including MOPS " also includes the conjugate base of MOPS.
As used herein, " coliform " refers to the microorganism for the coliform for belonging to bacterium, be used to indicate food, water and
The hygienic quality of beverage.Such bacterium is included into be defined lactose fermentation at the ability of acid and gaseous product by them.Large intestine
The non-limiting example of bacterium include be classified as citric acid Bacillus (Citrobacter), Enterobacter (Enterobacter),
Hafnia (Hafnia), Klebsiella (Klebsiella), Serratia (Serratia) and Escherichia
(Escherichia) strain.
The features and advantages of the present invention will pass through the detailed description and the appended claims of consideration preferred embodiment
And understood.These and other features of the invention and excellent are described in conjunction with the various illustrative embodiments of the present invention as follows
Point.
The foregoing invention content of the present invention is not intended to the disclosed embodiment of each of description present invention or each implementation
Mode.Exemplary implementation scheme is more particularly exemplified in the following drawings and specific implementation mode.By specific implementation mode,
Drawings and claims, other feature, target and advantage will become obvious.
Description of the drawings
Fig. 1 is an implementation of the method for the aerogenesis coliform for showing according to present invention statistics to be present in culture dairy products
The block diagram of scheme.
Specific implementation mode
Before explaining in detail any embodiment of the disclosure, it should be appreciated that the present invention is not limited only in its application
Structure detail mentioned in being illustrated below or shown in following Figure and distribution mode for components.The present invention can have other
Embodiment, and can be practiced or carried out in many ways.Furthermore, it is to be understood that wording used herein and art
Language is to be not construed as restrictive for illustration purposes."include", "comprise" herein or " having " and its modification make
With meaning to cover thereafter cited project and its equivalent form and additional project.It unless otherwise stated or limits, otherwise
Term " connection " and " connection " and its modification are pressed broad sense and are used, and cover the directly or indirectly connection of these two aspects and connection
It connects.In addition, " connection " and " connection " is not limited to physics or mechanical connection or connection.It should be appreciated that without departing from disclosure range
In the case of, other embodiments can be used and structure change or logic variation can be carried out.In addition, term such as " front ",
" rear portion ", " top ", " bottom " etc. are only used for describing element when element is related each other, but are not intended to the specific of description equipment
Be orientated, instruction or imply equipment necessary or required orientation or regulation invention as described herein how will use when in use,
Installation, display or positioning.
Coliform is used to indicate the hygienic quality of food and beverage (including such as milk and water).Although coliform is found in water
In raw environment, plant and soil environment, but their presence may indicate that the fecal pollution from warm-blooded animal.Coliform is usually led to
The ability that lactose fermentation is generated to acid and gas when they are cultivated at 35-37 DEG C is crossed, is detected in food or water sample.
Coliform includes the strain from multiple Pseudomonas, including such as citric acid Bacillus (Citrobacter), Enterobacter
(Enterobacter), Hafnia (Hafnia), Klebsiella (Klebsiella), Serratia
(Serratia) and Escherichia (Escherichia).
The method for counting coliform is known.The illustrative methods of statistics coliform are found in bacteriological analysis handbook
(Bacteriological Analytical Manual) (P.Feng et al.；2002；" coliform (Escherichia coli)
With statistics (the Enumeration of Escherichia coli and the of coliform (Coliform Bacteria)
Coliform Bacteria)”；Bacteriological analysis handbook (Bacteriological Analytical Manual), the 4th chapter；
The document, which is incorporated by reference, to be incorporated herein).This method include on agar medium or PETRIFILM dry can be again
It is hydrated the direct method of counting that test sample is cultivated in culture apparatus.This method further includes by cultivating survey in liquid medium
The Maximum probable number method (Most Probable Number, MPN) that test agent is estimated.Some regulatory authorities require detection breast
Aerogenesis in sugar is to confirm the strain of coliform.
It is currently known in the selective nutrient medium (example of at least one for cultivating the coliform in film culture apparatus
Such as purplish red cholate-lactose medium (Violet Red Bile-Lactose medium), " VRBL ") in, by certain coliforms
The gas that (such as serratia marcescens (Serratia marcescens)) lactose fermenters generate can contain culture dairy products
It is substantially reduced in sample.The method of the present invention is relied on using specific buffer solution to eliminate the culture dairy products in film culture apparatus
Inhibition effect, thus allow for the accurate statistics of coliform, or even still when the dairy products of culture are present in sample
So.
The disclosure is provided the coliform bacterium colony being present in the dairy products of culture using film culture apparatus statistics and forms list
The ameliorative way of position.This method is related to generating CO using MOPS buffer solutions to promote lactose fermentation2Gas.Surprisingly, with
Other similar strengths are compared with the buffer solution of pH, and MOPS buffer solutions provide for certain coliforms in the dairy products sample of culture to be changed
Kind aerogenesis.
Fig. 1 is to show that statistics is present in the coliform Colony Forming Unit in culture dairy products in film culture apparatus
The block diagram of one embodiment of ameliorative way.Method 100 includes forming the mixture of dairy products and diluent comprising culture
Step 10.In any embodiment, the dairy products of predetermined amount can be mixed with the diluent of predetermined volume to form mixture.
Therefore, the dairy products of culture dilute and be can record in diluent extension rate and use it for counting at the beginning of every gram or every milliliter
The coliform quantity (if any) of the dairy products of beginning culture.
In any embodiment, the extension rate of culture dairy products in the mixture can be for example, about 1:2, about 1:3,
About 1:4, about 1:5, about 1:10, about 1:100, about 1:1000, about 1:10000, or about 1:100000.
The dairy products of culture can be any one of the dairy products of a variety of cultures.The culture breast system detected using this method
The non-limiting example of product includes Yoghourt, cheese, sour cream, buttermilk, curdled milk, yoghurt, Calpis (calpis), opens
Fei Er (kefir), love blue (ayran), acidified milk (qatiq), yoghourt (clabber) and white soft junket.
Diluent for suspending and/or diluting food and beverage sample is known in the art, and includes for example steaming
Distilled water, buffer solution, saline solution, nutrient medium and their mixture.It is buffering according to the preferred diluent of the disclosure
Aqueous solution, it includes (3- (N- morpholines) propane sulfonic acid) (hereinafter " MOPS ").MOPS can in free acid form and/or MOPS
Free acid salts (such as sodium salt) are present in diluent.It is a kind of buffered aqueous solution according to the preferred diluent of the disclosure,
It includes certain density MOPS, by the coliform bacterium colony grown in film culture apparatus be effectively facilitated lactose fermentation at
Gas.In any embodiment, diluent may include >=20mM MOPS.In any embodiment, diluent may include≤
50mM MOPS.In any embodiment, diluent may include the about 20-50mM MOPS including end value.Imagine basis
Buffered aqueous solution of the disclosure comprising MOPS such as is used to promote the battalion of coliform growth optionally including other components
It supports substance and/or inhibits the selective agent of non-coliform growth.
In any embodiment, including the diluent of MOPS is before the dairy products with culture mix can have it is scheduled
pH.MOPS concentration and diluent pH in diluent will influence the pH of mixture.In any embodiment, predetermined pH can be
>5.In any embodiment, predetermined pH can be<8.In any embodiment, predetermined pH can be>5.In any embodiment
In, predetermined pH can be >=5.5 and≤7.5.In any embodiment, predetermined pH may be about 7.0.
Because diluent is mixed with the dairy products of culture, concentration of the concentration of MOPS than MOPS in diluent in mixture
It is lower.For example, when the diluent comprising 20mM MOPS is mixed with the dairy products of culture to form mixture, wherein cultivate
Dairy products carry out 1:5 dilute, a concentration of about 16mM of MOPS in mixture.Therefore, when the diluent comprising 50mM MOPS and training
When foster dairy products are mixed to form mixture, wherein dairy products carry out 1:5 dilutions, in mixture MOPS it is a concentration of about
40mM。
In any embodiment, mixture can be formed in container (such as dilution bottle, homogenizer bag).In any implementation
In scheme, can homogenized mix (such as using mixing apparatus known in the art) to form diluent distributed relatively uniformly, sample
Product and any microorganism (if being talked about present in it) so that microorganism can carry out accurate statistics.
In any embodiment, mixture is formed to may include forming the mixture with pH within a predetermined range.Example
Such as, in any embodiment, being formed, there is the mixture of pH within a predetermined range may include forming pH>5 mixture.
In any embodiment, being formed, there is the mixture of pH within a predetermined range may include forming pH<8 mixture.Any
In embodiment, the mixture that pH is 5.5 to 7.5 is formed.
Referring again to Fig. 1, method 100 further includes step 20, the step with mixture (that is, the dairy products comprising culture and
Diluent) to form hydrogel, the hydrogel includes the nutrient medium grown for coliform, has inoculation film culture apparatus
Imitate the MOPS of the lactose and effective concentration of concentration.In any embodiment, vaccinated film culture apparatus is formed at least to wrap
It includes and deposits to the mixture of predetermined volume in film culture apparatus.Film culture apparatus before inoculation may include giving birth to for coliform
Long nutrient medium and/or lactose.If the film culture apparatus before inoculation does not include nutrient medium and/or lactose, it
Can provide in the mixture (such as thinner composition) or they can be deposited into film culture apparatus and (such as pass through
Pipette), they can be mixed with mixture at this.
Such as with trade name PETRIFILMTMColiform/enumeration of coliforms plate (PETRIFILMTME.coli/
Coliform Count Plate) (product identification 6404, purchased from St. Paul, MN 3M companies (3M Company,
St.Paul, MN)), PETRIFILMTMEnumeration of coliforms plate (PETRIFILMTMColiform Count Plate) (product is compiled
Numbers 6410, it is purchased from 3M companies (3M Company)) and PETRIFILMTMHigh sensitivity enumeration of coliforms plate
(PETRIFILMTMHigh-Sensitivity Coliform Count Plate) (product identification 6405 is purchased from 3M companies (3M
Company)) the film culture apparatus sold is suitable for disclosed method.Aforementioned two kinds of film culture apparatus include breast
Sugar, the selective agent (bile salt) grown for the nutrient medium of coliform growth and the non-coliform of inhibition.Before inoculation, breast
Sugar, nutrient medium component and selective agent are substantially free of water.When rehydrated with the aqueous specimen of predetermined volume (such as when
When film culture apparatus is inoculated with the mixture of the dairy products comprising culture and diluent), hydrogel is formed in classification inoculation apparatus.
According to disclosed method, hydrogel includes the MOPS of the lactose and effective concentration of effective concentration.
The manufacture of film culture apparatus is described in such as United States Patent (USP) 4,565,783；5,089,413；5,137,812；With
In 5,232,838.Also a effective amount of MOPS or its salt can be mixed in such film culture apparatus before inoculation, such as international publication
Described in WO 2012/161992A1, the document, which is incorporated by reference, to be incorporated herein.Alternatively, as described in embodiment hereof,
MOPS can be provided in the diluent that the dairy products with culture mix.
Film culture apparatus may include that (such as device can be observed by it in base material (such as self-supporting base material), emulsion sheet
Internal emulsion sheet) and be placed between base material and emulsion sheet (such as in the coating) drying can be rehydrated can
It is dissolved in the gelling agent (such as guar gum, xanthans, locust bean gum or their mixture) of cold water.Optionally, thin before inoculation
Membrance cuiture device also may include nutriment, selective agent and/or be placed between base material and emulsion sheet (such as in the coating)
Indicator.
In any embodiment, the formation of vaccinated film culture apparatus includes the dairy products and dilute that will include culture
The mixture deposition (such as passing through pipette) of agent is released between the base material and emulsion sheet of film culture apparatus.As described above,
MOPS buffer solutions can be provided in the form of the component of film culture apparatus or thinner composition, as described herein in method 100
Step 10 in form mixture.Alternatively, it is contemplated that MOPS (such as in aqueous solution) can separately be deposited with mixture (such as it is logical
Cross pipette) in film culture apparatus, and then mixed with mixture, then it is closed vaccinated film culture apparatus.
It is contemplated that the formation of vaccinated film culture apparatus may also include will promote coliform growth nutriment and/
Or inhibit in the selective agent deposition (such as passing through pipette) to film culture apparatus of non-coliform growth.Nutriment and/or
Selective agent may be present in one or more solution, after they are deposited in film culture apparatus but be closed the device
Before, which can mix with mixture.
With may include coliform water-containing medium (such as mixture of diluent and the dairy products of culture) be inoculated with
Later, the cold-water-soluble gelling agent culture medium and hydrogel is formed in film culture apparatus.The hydrogel has
A certain amount of coliform can breed if there is in mixture to form bacterium colony in hydrogel, and the water that ferments
Lactose and thereby generation acid in gel and gaseous product.Be trapped in gas in the hydrogel close to each aerogenesis bacterium colony by
Observable bubble is formed in the hydrogel of nearly aerogenesis bacterium colony.Nutriment and/or selective agent, no matter they are before inoculation
It is present in film culture apparatus or deposits in film culture apparatus during inoculation, is diffused into hydrogel and forms battalion
Culture medium is supported, promotes coliform relative to the life for the other microorganisms (such as lactic acid bacteria) being likely to be present in culture dairy products
It is long.
When forming hydrogel after being inoculated with film culture apparatus, hydrogel is sandwiched between emulsion sheet and base material.
In any embodiment of this method, it includes being formed to be inoculated with film culture apparatus with the mixture for forming hydrogel
The hydrogel of MOPS with effective concentration.In any embodiment, the effective concentration of MOPS can be >=16mM in hydrogel.
In any embodiment, the effective concentration of MOPS can be≤40mM in hydrogel.In any embodiment, in hydrogel
The effective concentration of MOPS can be the 16-40mM including end value.
Include the water-setting to form the lactose with effective concentration with the mixture inoculation film culture apparatus of hydrogel is formed
Glue.The lactose of effective concentration is enough to allow the formation of observable bubble, and the bubble in film culture apparatus by growing
The lactose fermentation bacterium colony of coliform forms unit and generates.In any embodiment, the effective concentration of lactose can be in hydrogel
>=0.5% (w/v).In any embodiment, the effective concentration of lactose can be≤1.25% (w/v) in hydrogel.Any
In embodiment, the effective concentration of lactose can be 0.5% to≤1.25% (w/v) including end value in hydrogel.
Referring again to Fig. 1, after being inoculated with film culture apparatus with mixture, method 100 further includes promoting coliform life
The step 30 of vaccinated film culture apparatus is cultivated under conditions of length.Those skilled in the art, which will be recognized that, to be promoted
Vaccinated film culture apparatus is cultivated under conditions of coliform growth to may include cultivating at temperature more higher than environment temperature
Vaccinated film culture apparatus.Preferably, cultivate vaccinated film culture apparatus be included in it is more than or equal to 30 DEG C and small
In or equal to cultivating the culture apparatus at a temperature of 44 DEG C.In any embodiment, vaccinated film culture apparatus is cultivated,
The culture apparatus is cultivated at a temperature in the range of being included in 35-37 DEG C including end value.
In any embodiment, cultivating vaccinated film culture apparatus under conditions of promoting coliform growth includes
It cultivates the culture apparatus for a period of time, is enough to allow to be formed observable coliform bacterium colony.Form observable bacterium colony
Time can be about 4 hours to about 48 hours including end value；It preferably includes about 16 hours to about 26 small including end value
When；More preferably from about 18 to about 24 hours.It may include indicator (such as redox dye according to the film culture apparatus of the disclosure
Such as triphenyltetrazolium chloride；Such as the chloro- 3- indoles-β-D- galactopyranosides of the bromo- 4- of chromogenic enzyme substrate such as 5-)；Or
Fluorescent enzyme substrate (such as 4-methyl umbelliferone-β-D- galactopyranosides), reacts with coliform, result in colour developing and/
Or fluorescence-causing substance, this is conducive to bacterium colony observation.It is usually generated by the coliform bacterium colony grown in film culture apparatus considerable
The bubble observed accumulates in culture interior generation in 16-18 hours.During the culture period longer than 18 hours, bubble can continue to increase
Greatly to a certain degree
After cultivating vaccinated film culture apparatus for a period of time under conditions of promoting coliform growth, method 100 is also
The step 40 of row counting is dropped into including identification and to being present in the aerogenic bacteria in vaccinated culture apparatus.
Identify that the bacterium colony being present in vaccinated culture apparatus may include observing the water formed in film culture apparatus
Whether there is or not the instructions of bacterial clump for gel.Bacterium colony can be identified in film culture apparatus, such as pass through hydrogel as described above
Change colour to identify, the discoloration is associated with the bacterium in bacterium colony and the reaction between indicator.
Identify that the aerogenesis bacterium colony being present in vaccinated culture apparatus may include observing in hydrogel by biogenetic gas
Gap caused by bubble, the biogenesis bubble is instead of one be clipped between the base material and emulsion sheet of film culture apparatus
Divide hydrogel.Biogenesis bubble, which is generally near, generates its bacterium colony.The gap observed can be essentially colorless in hydrogel
High light transmittance region.Biogenesis bubble is observed in film culture apparatus is described in such as international publication WO 2014/
In 099644 A1, the document, which is incorporated by reference, to be incorporated herein.
By manufacturer's offer and PETRIFILM coliforms/enumeration of coliforms plate (ETRIFILM E.coli/
Coliform Count Plates) the deciphering guide (Interpretation Guide) that is used together provides about determination
The bubble observed in film culture apparatus guidance whether associated with specific bacterium colony.The guide portion be related to bubble with it is micro-
The proximity of biological bacterium colony.In general, contact specified microorganisms bacterium colony bubble be considered as it is associated with the bacterium colony (that is,
It is generated by the bacterium colony).In addition, bubble of its position in the distance equal to about three colony diameters is considered as and the bacterium colony phase
It is associated with (that is, being generated by the bacterium colony).Therefore, with 0.5mm diameters bacterium colony can generation position apart from bacterium colony most about 1.5mm's
Bubble.It is for determination relative to any bubble (such as the gap observed in hydrogel) position close to microbe colony
The no bubble is generated by the microorganism for forming the bacterium colony.
Gap in observation hydrogel may include gap of the observation with minimum dimension.In any embodiment, it observes
Gap in hydrogel includes observation along the gap for passing through the straight line in the gap to have at least size of 0.5mm.In any implementation
In scheme, it includes the gap that observation edge passes through the straight line in the gap to have at least size of 1.0mm to observe the gap in hydrogel.
In any embodiment, it includes the ruler that observation edge passes through the straight line in the gap to have most 3mm to observe the gap in hydrogel
Very little gap.
In any embodiment, identification and to be present in the aerogenic bacteria in vaccinated culture apparatus drop into row count can
Including identifying and being counted to coliform bacterium colony, with the coliform Colony Forming Unit phase being present in the dairy products of culture
Association.Therefore, the dairy products in culture are may indicate that there are aerogenesis bacterium colony in the film culture apparatus used in method of disclosure
In there are coliforms.
In any embodiment according to disclosed method, it includes manually to aerogenic bacteria that aerogenic bacteria, which is dropped into row and counted,
Drop into capable counting.As used herein manually to aerogenic bacteria drop into row count be included in the lower bacterium colony counting of instrument auxiliary (such as
Quebec colony counter (Quebec Colony Counter)), the illumination and/or amplification of film culture apparatus are provided.Separately
Selection of land, in any embodiment of method of disclosure, it includes using automatic bacterial colony counting equipment that aerogenic bacteria, which is dropped into row and counted,
Such as the PETRIFILM plate reading machines of the 3M companies (3M Company, St.Paul, MN) purchased from St. Paul, MN
(PETRIFILM Plate Reader) drops into capable counting to aerogenic bacteria.
It can be used for detecting certain strains of coliform according to disclosed method, wherein generation gas final product (such as
CO2) lactose fermentation in film culture apparatus by cultivating dairy products present in the sample for inoculated and cultured device
Inhibit.The non-limiting example of such coliform include liquefied Serratia (Serratia liquefaciens) strain at
Member.Other coliforms that its lactose fermentation aerogenesis can be inhibited by culture dairy products include enterobacter agglomerans
The member of (Enterobacter agglomerans) (also referred to as pantoea agglomerans (Pantoea agglomerans)) strain.
Embodiment described above and shown in the drawings is only presented by way of example, rather than is intended as general to the present invention
Read the limitation with principle.Therefore, it will be understood by those of ordinary skill in the art that, in the feelings for not departing from disclosure spirit and scope
Element and its construction and arrangement can be variously modified under condition.
All references cited herein and publication are clearly incorporated in a manner of being cited in full text in the disclosure.
Following embodiments is intended to illustrate the disclosure rather than be limited.
Exemplary implementation scheme
Embodiment A statistics is present in the method for the aerogenesis coliform in the dairy products of culture, the method includes：
A kind of mixture is formed, the mixture includes the diluent of the dairy products and predetermined volume of predetermined amount；
It is inoculated with film culture apparatus with the mixture to form hydrogel, the hydrogel includes the battalion grown for coliform
Support the MOPS of culture medium, the lactose of effective concentration and effective concentration；
Vaccinated film culture apparatus is cultivated under conditions of promoting coliform growth；And
It identifies and drops into capable counting to being present in the aerogenic bacteria in vaccinated culture apparatus.
Embodiment B is method according to embodiment A, wherein in the hydrogel MOPS effective concentration >=
16mM。
Embodiment C is method according to embodiment B, wherein in the hydrogel MOPS effective concentration≤
40mM。
Embodiment D is the method according to any one of foregoing embodiments, wherein the diluent includes MOPS.
Embodiment E is method according to embodiment D, wherein in the diluent MOPS concentration >=20mM.
Embodiment F is method according to embodiment E, wherein in the diluent MOPS concentration≤50mM.
Embodiment G is method according to embodiment F, wherein in the diluent concentration >=25mM of MOPS and
≤45mM。
Embodiment H is the method according to any one of foregoing embodiments, wherein the pH of the mixture>5.
Embodiment I is the method according to any one of foregoing embodiments, wherein the pH of the mixture<8.
Embodiment J is the method according to embodiment I, wherein pH >=5.5 of the mixture and≤7.5.
Embodiment K be culture dairy products, selected from Yoghourt, sour cream, buttermilk, curdled milk, yoghurt, can you must
Think (calpis), Kefir grains (kefir), love blue (ayran), acidified milk (qatiq), yoghourt (clabber) and white soft junket.
Embodiment L is the method according to any one of foregoing embodiments, wherein lactose in the hydrogel
Effective concentration >=0.5% (w/v).
Embodiment M is method according to embodiment L, wherein in the hydrogel lactose effective concentration≤
1.25% (w/v).
Embodiment N is the method according to any one of preceding claims, wherein dropping into capable counting to aerogenic bacteria
Include being counted to the bacterium colony of Serratia (Serratia).
Embodiment O is the method according to any one of foregoing embodiments, wherein being inoculated with the film culture
After device, the hydrogel includes the nutrient medium for promoting coliform growth.
Embodiment P is the method according to embodiment O, wherein before being inoculated with the film culture apparatus, institute
State at least one nutriment that culture apparatus includes the nutrient medium.
Embodiment Q is the method according to embodiment P, wherein at least one nutriment is as substantially
Water-free composition is present in the film culture apparatus.
Embodiment R is the method according to any one of foregoing embodiments, wherein being inoculated with the film culture
After device, the hydrogel includes the selective agent for inhibiting non-coliform growth.
Embodiment S is the method according to embodiment N, wherein before being inoculated with the film culture apparatus, institute
It includes the selective agent to state culture apparatus.
Embodiment T is the method according to embodiment S, wherein before being inoculated with the film culture apparatus, institute
Selective agent is stated as the composition substantially free of water to be present in the film culture apparatus.
Embodiment U is the method according to any one of foregoing embodiments, wherein promoting coliform growth
Under the conditions of cultivate the vaccinated film culture apparatus, be included at a temperature in the range of 35-37 DEG C including end value
Cultivate the film culture apparatus.
Embodiment V is the method according to any one of foregoing embodiments；The wherein described film culture apparatus packet
Include the cold-water-soluble gelling agent that can be rehydrated of base material, emulsion sheet and drying, the gelling agent setting in the base material and
Between the emulsion sheet；It includes the covering to form contact device to be wherein inoculated with film culture apparatus with mixture to form hydrogel
The hydrogel of piece and base material；And wherein identification aerogenesis bacterium colony includes the gap close to bacterium colony observed in hydrogel.
Embodiment W is the method according to embodiment V, wherein it includes observation to observe the gap in the hydrogel
Along the gap for passing through the straight line in the gap that there is at least size of 0.5mm.
Embodiment X is the method according to embodiment W, wherein the gap is arranged in the hydrogel, away from
Bacterial clump≤1.5mm visible from range estimation.
Embodiment Y is the method according to any one of preceding claims, wherein dropping into capable counting to aerogenic bacteria
Including dropping into capable counting to aerogenic bacteria manually.
Embodiment Z is the method according to any one of embodiment A to Y, wherein dropping into capable counting to aerogenic bacteria
Including using automatic bacterial colony counting equipment to drop into capable counting to aerogenic bacteria.
Embodiment
Reference implementation example 1-9：The incorporation liquefied Serratia being diluted in Butterfield (Butterfield) diluent The statistics of the culture dairy products of (Serratia liquefaciens)。
Film culture apparatus (PETRIFILM for these embodimentsTMHigh sensitivity enumeration of coliforms plate
(PETRIFILMTMHigh-Sensitivity Coliform Count Plates)) it is purchased from 3M companies (3M Company).Purchase
American Type Culture collection (American Type Culture from Virginia, USA Manassas
Collection, Manassas, VA) coliform (Serratia liquefaciens) (ATCC 51814) be used for this paper institutes
The experiment stated.Butterfield (Butterfield) diluent is obtained from the Edge of Tennessee State Memphis (Memphis, TN)
Biologicals companies.Different culture dairy products (as shown in table 1) are obtained from retail source.
A various dairy products are mixed with four parts of diluents (Butterfield (Butterfield) diluent) respectively, because
Total extension rate of the dairy products of this culture is 1:5.The overnight culture of liquefied Serratia (S.liquefaciens) bar
It is diluted in special Field (Butterfield) diluent, and the dilution culture of small size is added to containing diluted culture
To obtain the liquefied Serratia containing dilution dairy products and about 50 Colony Forming Unit (CFU) in each container of dairy products
(S.liquefaciens) mixture.The mixture is homogenized to spread more evenly across dairy products and bacterium in the mixture.
A part of mixture is removed to measure pH.
Each mixture takes one milliliter to be used to be inoculated with individual PETRIFILM according to the manufacturer's instructionsTMIt is high sensitive big
Intestinal flora tally (PETRIFILMTMHigh-Sensitivity Coliform Count Plates).After inoculation, 35
Tally is cultivated at DEG C about 24 hours.After incubation, the aerogenesis coliform in manual identification plate according to the manufacturer's instructions
Type bacterium colony, and record the clump count observed on each plate.
Reference pair is prepared as described in reference implementation example to shine, unlike, the dairy products without a culture and four parts of Barts
Field (Butterfield) diluent prepares the mixture of incorporation bacterium, but with five parts of Butterfields
(Butterfield) diluent prepares " mixture " (that is, the dairy products without culture in these samples.Reference pair, which is shone, to be shown in
It is able to observe that how many bacterium in the sample in the case of no any interference, which may be due to having culture in the sample
Dairy products and occur.
The aerogenesis clump count that table 2 is listed the pH of each reference implementation example and observed in each test sample.Data are aobvious
Show the liquefied Serratia (S.liquefaciens) of every milliliter of incorporation about 35CFU of each sample (referring to reference pair photograph).Data
Also show that each sample is added in the dairy products of culture is reduced to about 4.0-5.2 by the pH of sample mixture from about 7.8.In addition,
Data, which are shown in the CFU average ratios observed in the sample containing culture dairy products, has identical diluent but wherein without training
The reference pair for supporting dairy products shines low about 25CFU (that is, low about 70%).
Table 2：Contain the liquid in the sample of diluted culture dairy products in Butterfield (Butterfield) diluent
Change Serratieae (S.liquefaciens) to count.Every plate CFU averages in reference implementation example 1-9 are about 10.Each ginseng
The dairy products for examining the culture in embodiment are listed in table 1.The CFU of report indicates two same samples for each condition
Average value.
Comparative example 1-9：The incorporation liquefied Serratia being diluted in Butterfield (Butterfield) diluent The statistics of the culture dairy products of (Serratia liquefaciens)。
The film culture apparatus that is used in these embodiments, coliform, Butterfield (Butterfield) diluent and
The dairy products of culture are identical as those of being used in reference implementation example 1-9.Including the dairy products of culture, Butterfield
(Butterfield) diluent and the mixture of coliform are prepared as described in reference implementation example 1-9, unlike；It is trained in mixing
After foster dairy products and diluent, the 0.1N NaOH of small size (≤1mL) are added in mixture, thus by the pH of mixture
It is adjusted to about 7.0.
It is inoculated with as described in reference implementation example 1-9 and cultivates film culture apparatus.After the training period, such as reference implementation example 1-9
The identification simultaneously counts the aerogenesis bacterium colony in film culture apparatus.
It is prepared as described in comparative example 1-9 and compares control A, unlike, the dairy products without a culture and four parts of Barts
Field (Butterfield) diluent prepares the mixture of incorporation bacterium, but with five parts of Butterfields
(Butterfield) diluent prepares " mixture " (that is, the dairy products without culture in these samples.Compare control A to be shown in
It is able to observe that how many bacterium in the sample in the case of no any interference, which may be due to having culture in the sample
Dairy products and occur.
The aerogenesis clump count that table 3 is listed the pH of each comparative example and observed in each test sample.Data are shown often
The liquefied Serratia (S.liquefaciens) of every milliliter of incorporation about 35CFU of a sample (referring to control A is compared).Data are shown
The CFU average ratios observed in the sample containing culture dairy products have identical diluent but wherein without culture dairy products
The low about 11CFU of comparison control A (that is, low about 35%).
Table 3：Contain the culture dairy products that pH is diluted and then adjusted in Butterfield (Butterfield) diluent
Sample in liquefied Serratia (S.liquefaciens) statistics.Every plate CFU averages in comparative example 1-9 are about
19.The dairy products of culture in each comparative example are listed in table 1.The CFU of report indicate two for each condition it is identical
The average value of sample.
Comparative example 10-18：It is diluted to comprising the incorporation liquefied Serratia (Serratia in the phosphatic diluents of 20mM Liquefaciens the statistics of culture dairy products)。
In the dairy products and reference implementation example 1-9 of the film culture apparatus, coliform and the culture that are used in these embodiments
It those of uses identical.Diluent (20mM potassium phosphates, pH7.05) is prepared using SILVER REAGENT dipotassium hydrogen phosphate.Include the breast of culture
The mixture of product, phosphate-buffered diluent and coliform is prepared as described in reference implementation example 1-9.
It is inoculated with as described in reference implementation example 1-9 and cultivates film culture apparatus.After the training period, such as reference implementation example 1-9
The identification simultaneously counts the aerogenesis bacterium colony in film culture apparatus.
It is prepared as described in comparative example 10-18 and compares control B, unlike, without the dairy products of a culture and four parts
20mM phosphate-buffered diluents prepare the mixture of incorporation bacterium, but are prepared with five parts of 20mM phosphate-buffered diluents
" mixture " (that is, the dairy products without culture in these samples).Compare in the case that control B is shown in without any interference in sample
How many bacterium are able to observe that in product, which may be due to occurring in the presence of the dairy products of culture in the sample.
The aerogenesis clump count that table 4 is listed the pH of each comparative example and observed in each test sample.Data are shown often
The liquefied Serratia (S.liquefaciens) of every milliliter of incorporation about 29CFU of a sample (referring to control B is compared).Data are shown
The CFU average ratios observed in the sample containing culture dairy products have identical diluent but wherein without culture dairy products
The low about 15CFU of comparison control B (that is, low about 52%).
Table 4：Contain the liquefaction sand Lei Shi in the sample of diluted culture dairy products in 20mM phosphate-buffered diluents
Bacterium (S.liquefaciens) counts.Every plate CFU averages in comparative example 10-18 are about 14.Training in each comparative example
Foster dairy products are listed in table 1.The CFU of report indicates the average value of two same samples for each condition.
Comparative example 19-27：It is diluted to comprising the incorporation liquefied Serratia (Serratia in the phosphatic diluents of 35mM Liquefaciens the statistics of culture dairy products)。
In the dairy products and reference implementation example 1-9 of the film culture apparatus, coliform and the culture that are used in these embodiments
It those of uses identical.Diluent (35mM potassium phosphates, pH7.01) is prepared using SILVER REAGENT dipotassium hydrogen phosphate.Include the breast of culture
The mixture of product, phosphate-buffered diluent and coliform is prepared as described in reference implementation example 1-9.
It is inoculated with as described in reference implementation example 1-9 and cultivates film culture apparatus.After the training period, such as reference implementation example 1-9
The identification simultaneously counts the aerogenesis bacterium colony in film culture apparatus.
It is prepared as described in comparative example 19-27 and compares control C, unlike, without the dairy products of a culture and four parts
35mM phosphate-buffered diluents prepare the mixture of incorporation bacterium, but are prepared with five parts of 35mM phosphate-buffered diluents
" mixture " (that is, the dairy products without culture in these samples).Compare in the case that control C is shown in without any interference in sample
How many bacterium are able to observe that in product, which may be due to occurring in the presence of the dairy products of culture in the sample.
The aerogenesis clump count that table 5 is listed the pH of each comparative example and observed in each test sample.Data are shown often
The liquefied Serratia (S.liquefaciens) of every milliliter of incorporation about 33CFU of a sample (referring to control C is compared).Data are shown
The CFU average ratios observed in the sample containing culture dairy products have identical diluent but wherein without culture dairy products
The low about 21CFU of comparison control C (that is, low about 64%).
Table 5：Contain the liquefaction sand Lei Shi in the sample of diluted culture dairy products in 35mM phosphate-buffered diluents
Bacterium (S.liquefaciens) counts.Every plate CFU averages in comparative example 19-27 are about 12.Training in each comparative example
Foster dairy products are listed in table 1.The CFU of report indicates the average value of two same samples for each condition.
Embodiment 1-9：Incorporation liquefied Serratia (the Serratia being diluted in the diluent comprising 20mM MOPS Liquefaciens the statistics of culture dairy products)。
In the dairy products and reference implementation example 1-9 of the film culture apparatus, coliform and the culture that are used in these embodiments
It those of uses identical.Diluent (20mM MOPS, pH7.04) is prepared using SILVER REAGENT MOPS free acids and MOPS sodium salts.Packet
Dairy products containing culture, the diluent comprising MOPS and the mixture of coliform are prepared as described in reference implementation example 1-9.
It is inoculated with as described in reference implementation example 1-9 and cultivates film culture apparatus.After the training period, such as reference implementation example 1-9
The identification simultaneously counts the aerogenesis bacterium colony in film culture apparatus.
Embodiment control 1 is prepared as described in embodiment 1-9, unlike, without the dairy products of a culture and four parts
20mM MOPS diluents prepare incorporation bacterium mixture, but with five parts of 20mM MOPS diluents prepare " mixture " (that is,
The dairy products without culture in these samples).Embodiment control 1 can be seen in the sample in the case of being shown in without any interference
How many bacterium are observed, which may be due to occurring in the presence of the dairy products of culture in the sample.
The aerogenesis clump count that table 6 is listed the pH of each embodiment and observed in each test sample.Data are shown often
The liquefied Serratia (S.liquefaciens) (compareing 1 referring to embodiment) of every milliliter of incorporation about 29CFU of a sample.Data are aobvious
Show that the CFU average ratios observed in the sample containing culture dairy products have identical diluent but wherein without culture breast system
The embodiment of product compares 1 only low about 7CFU (that is, low about 24%).
Table 6：Contain the liquefied Serratia in the sample of diluted culture dairy products in 20mM MOPS buffered diluents
(S.liquefaciens) it counts.Every plate CFU averages in embodiment 1-9 are about 22.Culture in each embodiment
Dairy products are listed in table 1.The CFU of report indicates the average value of two same samples for each condition.
Embodiment 10-18：Incorporation liquefied Serratia (the Serratia being diluted in the diluent comprising 35mM MOPS Liquefaciens the statistics of culture dairy products)。
In the dairy products and reference implementation example 1-9 of the film culture apparatus, coliform and the culture that are used in these embodiments
It those of uses identical.Diluent (35mM MOPS, pH7.03) is prepared using SILVER REAGENT MOPS free acids and MOPS sodium salts.Packet
Dairy products containing culture, the diluent comprising MOPS and the mixture of coliform are prepared as described in reference implementation example 1-9.
It is inoculated with as described in reference implementation example 1-9 and cultivates film culture apparatus.After the training period, such as reference implementation example 1-9
The identification simultaneously counts the aerogenesis bacterium colony in film culture apparatus.
Embodiment control 2 is prepared as described in embodiment 10-18, unlike, without the dairy products of a culture and four parts
35mM MOPS diluents prepare incorporation bacterium mixture, but with five parts of 35mM MOPS diluents prepare " mixture " (that is,
The dairy products without culture in these samples).Embodiment control 2 can be seen in the sample in the case of being shown in without any interference
How many bacterium are observed, which may be due to occurring in the presence of the dairy products of culture in the sample.
The aerogenesis clump count that table 7 is listed the pH of each embodiment and observed in each test sample.Data are shown often
The liquefied Serratia (S.liquefaciens) (compareing 2 referring to embodiment) of every milliliter of incorporation about 46CFU of a sample.Data are aobvious
Show that the CFU average ratios observed in the sample containing culture dairy products have identical diluent but wherein without culture breast system
The embodiment of product compares 2 only low about 4CFU (that is, low about 9%).
Table 7：Contain the liquefied Serratia in the sample of diluted culture dairy products in 35mM MOPS buffered diluents
(S.liquefaciens) it counts.Every plate CFU averages in embodiment 10-18 are about 42.Culture in each embodiment
Dairy products listed in table 1.The CFU of report indicates the average value of two same samples for each condition.
The disclosures of all patents, the complete disclosure of patent application and publication and available electronic edition
Material is herein incorporated by reference.In the public affairs of present disclosure and any document being hereby incorporated herein by
Open between content there are it is any it is inconsistent in the case of, should be subject to present disclosure.Foregoing detailed description and implementation
Example provides only for the present invention is expressly understood.But they are understood not to unnecessary limitation.The present invention is not limited to show
Detail go out and description, obvious variations are included within for those skilled in the art is wanted by right
It asks in the present invention defined by book.
All titles are and to should not be taken to limit the meaning of the text of the header in order to which reader is convenient, are removed
Be far from it regulation.
Under the premise of not departing from the spirit and scope of the invention, various modifications can be made.These and other embodiment
In the scope of the following claims.
Claims (20)
1. a kind of statistics is present in the method for the aerogenesis coliform in the dairy products of culture, the method includes：
A kind of mixture is formed, the mixture includes the diluent of the dairy products and predetermined volume of predetermined amount；
It is inoculated with film culture apparatus with the mixture to form hydrogel, the hydrogel includes the nutrition grown for coliform
The MOPS of culture medium, the lactose of effective concentration and effective concentration；
The vaccinated film culture apparatus is cultivated under conditions of promoting coliform growth；And
It identifies and drops into capable counting to being present in the aerogenic bacteria in the vaccinated culture apparatus.
2. according to the method described in claim 1, in the wherein described hydrogel MOPS effective concentration >=16mM.
3. according to the method described in claim 2, in the wherein described hydrogel MOPS effective concentration≤40mM.
4. according to any method of the preceding claims, wherein the diluent includes the MOPS.
5. according to the method described in claim 4, in the wherein described diluent MOPS concentration >=20mM.
6. according to the method described in claim 5, in the wherein described diluent MOPS concentration >=25mM and≤45mM.
7. according to any method of the preceding claims, wherein the pH of the mixture is between 5.0 and 8.0.
8. according to any method of the preceding claims, wherein the dairy products of the culture be selected from Yoghourt, sour cream,
Buttermilk, curdled milk, yoghurt, Calpis, Kefir grains, love orchid, acidified milk, yoghourt and white soft junket.
9. according to any method of the preceding claims, wherein in the hydrogel lactose effective concentration >=
0.5% (w/v).
10. according to the method described in claim 9, in the wherein described hydrogel lactose effective concentration≤1.25% (w/v).
11. according to any method of the preceding claims, wherein it includes to husky Lei Shi that aerogenic bacteria, which is dropped into row and counted,
The bacterium colony of Pseudomonas is counted.
12. described according to any method of the preceding claims, wherein after being inoculated with the film culture apparatus
Hydrogel includes the nutrient medium for promoting coliform growth.
13. described according to any method of the preceding claims, wherein after being inoculated with the film culture apparatus
Hydrogel includes the selective agent for inhibiting non-coliform growth.
14. according to the method for claim 13, wherein before being inoculated with the film culture apparatus, it is being inoculated with the film
Before culture apparatus, the culture apparatus includes the selective agent.
15. according to the method for claim 14, wherein the selective agent is present in as the composition substantially free of water
In the film culture apparatus.
16. according to any method of the preceding claims, wherein being cultivated under conditions of promoting coliform growth
The film culture apparatus of inoculation, cultivates the film at a temperature in the range of being included in 35-37 DEG C including end value
Culture apparatus.
17. according to any method of the preceding claims；The wherein described film culture apparatus includes base material, emulsion sheet
With dry cold-water-soluble gelling agent that can be rehydrated, gelling agent setting the base material and the emulsion sheet it
Between；It includes forming contact described device to be wherein inoculated with the film culture apparatus with the mixture to form the hydrogel
The hydrogel of the emulsion sheet and the base material；And wherein identification aerogenesis bacterium colony includes connecing in the observation hydrogel
The gap of the nearly bacterium colony.
18. according to the method for claim 17, wherein it includes that observation is described along passing through to observe the gap in the hydrogel
The straight line in gap has the gap of at least size of 0.5mm.
19. according to the method for claim 18, wherein the gap is arranged in the hydrogel, distance is estimated visible
Bacterial clump≤1.5mm.
20. according to any method of the preceding claims, wherein it includes using automatic that aerogenic bacteria, which is dropped into row and counted,
Bacterium colony counting equipment drops into capable counting to the aerogenic bacteria.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201562263932P | 2015-12-07 | 2015-12-07 | |
| US62/263,932 | 2015-12-07 | ||
| PCT/US2016/065044 WO2017100143A1 (en) | 2015-12-07 | 2016-12-06 | Method of enumerating coliform colonies |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108368533A true CN108368533A (en) | 2018-08-03 |
Family
ID=57708741
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201680071402.8A Pending CN108368533A (en) | 2015-12-07 | 2016-12-06 | The method for counting big visible peristalsis visible intestinal peristalsis bacterium colony |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US10662460B2 (en) |
| EP (1) | EP3387144B1 (en) |
| JP (2) | JP7136407B2 (en) |
| CN (1) | CN108368533A (en) |
| WO (1) | WO2017100143A1 (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6306621B1 (en) * | 1991-11-18 | 2001-10-23 | The United States Of America As Represented By The Administrator Of The U.S. Environmental Protection Agency | Membrane filter agar medium for simultaneous detection of total coliforms and E. coli |
| CN101952457A (en) * | 2007-12-21 | 2011-01-19 | 3M创新有限公司 | The department of microbiology of the fluid sample analysis method of unifying |
| CN102325894A (en) * | 2008-12-31 | 2012-01-18 | 3M创新有限公司 | Coliform detection process and kit for use therein |
| CN103547678A (en) * | 2011-05-20 | 2014-01-29 | 3M创新有限公司 | Salmonella detection articles and methods of use |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4565783A (en) | 1981-01-27 | 1986-01-21 | Minnesota Mining And Manufacturing Company | Dry culture media |
| US5089413A (en) | 1989-05-19 | 1992-02-18 | Minnesota Mining And Manufacturing Company | Method and apparatus for culturing with microbiological dry culture medium |
| CA2022614A1 (en) | 1989-08-31 | 1991-03-01 | Richard R. Matner | Colony blotting method and device |
| US5232838A (en) | 1991-12-09 | 1993-08-03 | Minnesota Mining And Manufacturing Company | Culture media device and method of use |
| US5364766A (en) | 1993-05-14 | 1994-11-15 | Minnesota Mining And Manufacturing Company | Culture medium for rapid count of coliform bacteria |
| US8298758B2 (en) * | 2003-12-26 | 2012-10-30 | Prima Meat Packers, Ltd. | Method of multiplex microorganism detection |
| BR112015014821A2 (en) | 2012-12-20 | 2017-07-11 | 3M Innovative Properties Co | computer readable method and media |
-
2016
- 2016-12-06 US US15/781,547 patent/US10662460B2/en active Active
- 2016-12-06 WO PCT/US2016/065044 patent/WO2017100143A1/en active Application Filing
- 2016-12-06 JP JP2018549134A patent/JP7136407B2/en active Active
- 2016-12-06 EP EP16820403.0A patent/EP3387144B1/en active Active
- 2016-12-06 CN CN201680071402.8A patent/CN108368533A/en active Pending
-
2021
- 2021-07-08 JP JP2021113207A patent/JP7156763B2/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6306621B1 (en) * | 1991-11-18 | 2001-10-23 | The United States Of America As Represented By The Administrator Of The U.S. Environmental Protection Agency | Membrane filter agar medium for simultaneous detection of total coliforms and E. coli |
| CN101952457A (en) * | 2007-12-21 | 2011-01-19 | 3M创新有限公司 | The department of microbiology of the fluid sample analysis method of unifying |
| CN102325894A (en) * | 2008-12-31 | 2012-01-18 | 3M创新有限公司 | Coliform detection process and kit for use therein |
| CN103547678A (en) * | 2011-05-20 | 2014-01-29 | 3M创新有限公司 | Salmonella detection articles and methods of use |
Non-Patent Citations (2)
| Title |
|---|
| M.P.SILVA ET AL.: ""Coliforms at 45ºc and comparison of most probable number method and Petrifilm EC for enumeration of total coliforms and Escherichia coli of foods"", 《CIENC. TECNOL. ALIMENT., CAMPINAS》 * |
| RONALD M. ATLAS: "《Handbook of microbiological media Fourth Edition》", 31 December 2010, CRC PRESS * |
Also Published As
| Publication number | Publication date |
|---|---|
| EP3387144B1 (en) | 2020-03-25 |
| US20180355398A1 (en) | 2018-12-13 |
| WO2017100143A1 (en) | 2017-06-15 |
| JP7136407B2 (en) | 2022-09-13 |
| JP2021176309A (en) | 2021-11-11 |
| JP7156763B2 (en) | 2022-10-19 |
| US10662460B2 (en) | 2020-05-26 |
| EP3387144A1 (en) | 2018-10-17 |
| JP2018536437A (en) | 2018-12-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5601998A (en) | Culture medium and device for detection and enumeration of enterobacteriaceae | |
| CN104105795B (en) | The method for detecting Salmonella microorganisms | |
| EP2918672B1 (en) | Method for determining the sensitivity of microorganisms to antimicrobial substances | |
| KR101548513B1 (en) | Method and medium for detecting the presence or absence of staphylococcus aureus in a test sample | |
| CN110055301A (en) | A method of the culture medium of detection Bifidobacterium and quickly detection count | |
| CN106556599B (en) | A kind of method for rapid inspecting animalcule | |
| JP5737947B2 (en) | Method for detecting and / or identifying Clostridium difficile | |
| CN108486213A (en) | The method for detecting Salmonella microorganisms | |
| CN101717813B (en) | Method for detecting total number of bacterial colonies in activated lactobacillus drink | |
| US20100297692A1 (en) | Reaction medium for detecting and/or identtifying staphyloccous aureus | |
| CN101935688B (en) | Method for detecting and counting live microbes | |
| CN110475870B (en) | Fast detection of E.coli in a thin film culture apparatus | |
| CN108368533A (en) | The method for counting big visible peristalsis visible intestinal peristalsis bacterium colony | |
| CN106755275A (en) | Culture medium and its application of B group streptococcus β Hemolytic Types and γ Hemolytic Type bacterial strains are detected simultaneously | |
| CN103614453A (en) | Gastrointestinal bacteria detection method for medical microbiology experiment | |
| CN106086159B (en) | A kind of zymolyte culture medium that can detect two kinds of fecal pollution indicator bacterias simultaneously and its application | |
| CN107287275B (en) | Culture medium, kit containing culture medium and application of culture medium | |
| CN105200116B (en) | A kind of method of Paenibacillus polymyxa content in quick measure complex micro organism fungicide | |
| JP2012217440A (en) | Bag-shaped vessel for detecting microorganism | |
| CN104745671A (en) | Determination method of content of Escherichia coli for resisting interference of lactic acid zymophyte | |
| CN100430486C (en) | Quick determination for microbe munity | |
| CN103403178B (en) | Include the microbiological culture media of p-aminobenzoic acid alternatively agent | |
| Keddis et al. | Microbiology laboratory exercises | |
| CN106811403A (en) | A kind of test piece of quick detection bacillus cereus and preparation method thereof, detection method | |
| RU2571940C1 (en) | Method of culturing diphtheria bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |